The present invention relates generally to chelation compounds, radionuclide metal chelate compounds (i.e., complexes) and radiolabeled targeting moieties (i.e., conjugates) formed therefrom, and methods of making and using these compounds, complexes and conjugates for diagnostic and therapeutic purposes. This invention is more particularly related to compounds in which at least two of the chelating atoms are nitrogen atoms which are directly attached to aromatic rings and there is a non-hydrogen substituent directly attached to at least one of these nitrogen chelating atoms.
Radiolabeled chelation compounds have been studied and used as pharmaceuticals for diagnostic and therapeutic purposes for a number of years. The requirements for a useful radiolabeled chelating compound are well known to those skilled in the art of nuclear medicine and radiopharmaceutical research. Briefly, these requirements include: efficient final preparation of the radiopharmaceutical, such that preparation in the hospital or pharmacy is possible; efficient transport of the radiopharmaceutical to the target organ; efficient extraction of the radiopharmaceutical by the target organ, such that adequate target to background ratios are achieved to allow diagnostic and therapeutic distinctions; and adequate retention in the target organ to allow detection and therapy using conventionally available radiation monitoring equipment. Representative organs of interest are those containing malignant cells or activated platelets. Imaging agents and therapeutic agents have typically been unsuitable due to poor in vivo stability post-chelation, resulting in inadequate retention and accretion by the effected cells.
Thus, there is a need in the art for improved chelation compounds for imaging and therapy. The present invention fulfills this need and further provides other related advantages.
Briefly stated, the present invention in one aspect provides compounds having.the formula: 
wherein:
n=0 or 1;
R1 and R2 are independently selected from hydrogen, xe2x95x90O, with the proviso that both are not xe2x95x90O, xe2x80x94(CH2)mxe2x80x94Z where m is 0-10 and Z represents a conjugation group or targeting moiety, and xe2x80x94(Ch2)mxe2x80x94W where m is 0-10 and W represents a hydrolyzable group, or R1 and R2 are taken together to form a cyclic anhydride or a benzene ring;
R3 is hydrogen, lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z or xe2x80x94(CH2)mxe2x80x94W;
R4 and R5 are attached at one or more of the ring positions and are independently selected from hydrogen, lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z and xe2x80x94(CH2)mxe2x80x94W;
R6 and R7 are independently selected from hydrogen with the proviso that both are not hydrogen, lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z, xe2x80x94(CH2)mxe2x80x94W and 
xe2x80x83where Q represents a multivalent acid functionality group able to coordinate with metal ions, and p=0 to 1; R12 and R13 are independently selected from hydrogen, hydroxyl, carboxyl, phosphonic and hydrocarbon radicals having from 1-10 carbon atoms, and physiologically acceptable salts of the acid radicals;
X, Xxe2x80x2, Y and Yxe2x80x2 are independently selected from carbon, nitrogen, oxygen and sulfur to independently form five or six member aromatic rings wherein the remaining ring atoms are carbon;
A and Axe2x80x2 are independently selected from sulfur, nitrogen and oxygen, where sulfur may bear a hydrogen or a sulfur protecting group, or where A and Axe2x80x2 are both sulfur, A and Axe2x80x2 may be joined together by a bond; where an oxygen or a nitrogen may bear a hydrogen; or where A or Axe2x80x2 is nitrogen, A may bear R8 or R10 or both and Axe2x80x2 may bear R9 or R11 or both, wherein R8, R9, R10 and R11 are independently selected from lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z, xe2x80x94(CH2)mxe2x80x94W and 
xe2x80x83or R8 and R10 may be joined to form a cyclic anhydride or R9 and R11 may be joined to form a cyclic anhydride; or when A and Axe2x80x2 are both nitrogen, R10 and R11 may be joined to form T, where T 
xe2x80x83and n is 0 to 1, and R1xe2x80x2 and R2xe2x80x2 are independently selected from hydrogen, xe2x95x90O, with the proviso that both are not xe2x95x90O, xe2x80x94(CH2)mxe2x80x94Z and xe2x80x94(CH2)mxe2x80x94W or R1xe2x80x2 and R2xe2x80x2 are taken together to form a cyclic anhydride or a benzene ring; and R3xe2x80x2 is hydrogen, lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z and xe2x80x94(CH2)mxe2x80x94W; and
said compound has at least.one Z, W and Q.
In another aspect, the invention provides chelates comprising radionuclide metals (including oxides or nitrides thereof) complexed by a compound described above. A preferred metal chelate compound is of the formula: 
wherein:
M is a radionuclide metal or an oxide or a nitride thereof selected from technetium, copper, rhenium, sumarian, yttrium, indium, lead, bismuth, ruthenium, rhodium, gold and palladium;
n=0 or 1;
R1 and R2 are independently selected from hydrogen, xe2x95x90O with the proviso that both are not xe2x95x90O, xe2x80x94(CH2)mxe2x80x94Z where m is 0-10 and Z represents a conjugation group or targeting moiety, and xe2x80x94(CH2)mxe2x80x94W where m is 0-10 and W represents a hydrolyzable group, or R1 and R2 are taken together to form a cyclic anhydride or a benzene ring;
R3 is hydrogen, lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z or xe2x80x94(CH2)mxe2x80x94W;
R4 and R5 are attached at one or more of the ring positions and are independently-selected from hydrogen, lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z and xe2x80x94(CH2)mxe2x80x94W;
R6 and R7 are independently selected from hydrogen with the proviso that both are not hydrogen, lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z, xe2x80x94(CH2)mxe2x80x94Wxe2x80x94 and 
xe2x80x83where Q represents a multivalent acid functionality group able to coordinate with metal ions, and p=0 to 1; R12 and R13 are independently selected from hydrogen, hydroxyl, carboxyl, phosphonic and hydrocarbon radicals having from 1-10 carbon atoms, and physiologically acceptable salts of the acid radicals;
X, Xxe2x80x2, Y and Yxe2x80x2 are independently selected from carbon, nitrogen, oxygen and sulfur to independently form 5 or 6 member aromatic rings wherein the remaining ring atoms are carbon;
A and Axe2x80x2 are independently selected from sulfur, nitrogen and oxygen, where A or Axe2x80x2 is nitrogen, A may bear R8 or R10 or both and Axe2x80x2 may bear R9 or R11 or both, wherein R8, R9, R10 and R11 are independently selected from lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z, xe2x80x94(CH2)mxe2x80x94W and 
or R8 and R10 may be joined to form a cyclic anhydride or R9 and R11 may be joined to form a cyclic anhydride; or when A and Axe2x80x2 are both nitrogen, R10 and R11 may be joined to form T, where T is 
xe2x80x83and n is 0 to 1, and R1xe2x80x2 and R2xe2x80x2 are independently selected from hydrogen, xe2x95x90O, with the proviso that both are not xe2x95x90O, xe2x80x94(CH2)mxe2x80x94Z or R1xe2x80x2 and R2xe2x80x2 are taken together to form a cyclic anhydride or a benzene ring; and R3xe2x80x2 is hydrogen, lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z or xe2x80x94(CH2)mxe2x80x94W; and
said compound has at least one Z, W or Q.
Yet another aspect of the invention provides for the use of the chelation compounds described above in methods for diagnostic and therapeutic purposes. A diagnostic method is described for detecting the presence or absence of a target site within a mammalian host. This method comprises providing to cells a diagnostically effective dose of a compound of the present invention which contains a metal radionuclide, such as 99mTc and/or 111In, and detecting the biodistribution of the radionuclide. A therapeutic method is described for delivering a radionuclide, such as 186Re/188Re, 90Y, and 153Sm, to a target site within a mammalian host. This method comprises providing to cells a therapeutically effective dose of a chelate compound of the present invention.
Other aspects of the invention will become evident upon reference to the following detailed description.
Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter.
Targeting moietyxe2x80x94is any molecule that binds to a defined population of cells, and includes analogs of naturally occurring and synthetically or recombinantly prepared molecules. The targeting moiety may bind a receptor, an oligonucleotide, an enzymatic substrate, an antigenic determinant, or other binding site present on or in the target cell population. For example, a protein may be a targeting moiety. Antibodies and peptides are used throughout the specification as prototypical examples of targeting moieties. Tumor is used as a prototypical example of a target in describing the present invention.
Proteinxe2x80x94as used herein, includes proteins, fusion proteins, polypeptides and peptides; and may be an intact molecule, a fragment thereof, or a functional equivalent thereof; and may be genetically engineered.
Antibodyxe2x80x94as used herein, includes both polyclonal and monoclonal antibodies; and may be an intact molecule, a fragment thereof, or a functional equivalent thereof; and may be genetically engineered; examples of antibody fragments include F(abxe2x80x2)2, Fabxe2x80x2, Fab and Fv.
The present invention provides chelation compounds and radionuclide metal chelate compounds (i.e., complexes) prepared therefrom, as well as radiolabeled targeting moieties having the chelation compounds or chelates attached thereto (i.e., conjugates). The radionuclide metal chelates of the present invention may be attached to targeting moieties, such as antibodies and proteins, to form radiolabeled targeting moieties having diagnostic and therapeutic use. Alternatively, the radionuclide metal chelates of the present invention may be used for diagnostic and therapeutic purposes without attachment to targeting moieties.
The present invention provides compounds that have a variety of uses, including for malignant cell imaging and therapy as well as thrombus imaging. The compounds are capable of rapidly complexing a metal as well as forming a stable metal chelate (complex). The presence of nitrogen atoms within the chelating compound accelerates complex formation with the metal. This acceleration is due in part to the fact that a metal (e.g., technetium) is a soft acid, and nitrogen (in the form of an amine or amide) is a base. Amines generally provide for a greater increase in chelation rates than amides. Where sulfur atoms are additionally present within the chelating compound, they also provide for an increased rate of metal complexation and contribute to the stability of the resulting chelate. The presence of phenolic hydroxyl groups within the chelating compound aid in faster kinetics of metal ion chelation. The compounds of the present invention are characterized by desirable metal complex formation kinetic properties and desirable metal-chelate retention thermodynamic properties. The compounds of the present invention have the further advantage of nitrogen atoms attached directly to aromatic rings which aid in fast kinetics of chelation and further enhances the stability of the aromatic esters of this invention with respect to hydrolysis in the bloodstream. Furthermore, an additional advantage of the present invention is the presence of substituents attached to the nitrogen atoms within the chelating compound, which imparts a higher basicity to the chelation compound and allows for additional donor atoms for complexation, thereby expanding the type of radionuclides useful for radiotherapy and radiodetection in the present invention. In addition to the above advantages, the presence of substituents enhances pharmakokinetics and pharmacodynamics, such as the biopharmaceutical properties (i.e., absorption, distribution, metabolism and excretion).
The chelation compounds of the present invention have the following formula (I): 
Examples of specific embodiments of the elements of the above formula include the following.
R1 and R2 may be independently selected from hydrogen (H); an oxy group (xe2x95x90O); xe2x80x94(CH2)mxe2x80x94Z where m is 0-10 and Z represents a conjugation group or targeting moiety; or xe2x80x94(CH2)mxe2x80x94W where m is 0-10 and W represents a hydrolyzable group. As used 3 herein, the phrase independently selected means the selection of one substituent may be made without regard for the selection of any other substituent. Alternatively, R1 and R2 may be taken together to form a cyclic group, such as an anhydride or a benzene ring. As used herein, a benzene ring may be benzene or benzene with one or more substituents. A substituent may be any electron donating (methyl, methoxy, amino and the like) and/or electron withdrawing (halogens, nitro, carboxy, nitrile and the like) and functional groups (esters, imidates, carbaminates and the like) known in the art. Examples of such substituents include Cl, CH3, OCH3, F, Br, I, CF3 and a triazene, such a xe2x80x94Nxe2x95x90Nxe2x80x94N(CH3)2.
As noted above, Z represents a conjugation group or a targeting moiety. A xe2x80x9cconjugation groupxe2x80x9d in the compounds of the present invention is any chemically reactive group capable of forming a covalent bond with a targeting moiety under conditions that do not adversely affect the targeting moiety""s functional properties. For example, where the targeting moiety is a protein such as an antibody, the conjugation group is sufficiently reactive with a functional group on the protein so that the reaction can be conducted in substantially aqueous solutions and does not have to be forced (e.g., by heating to high temperatures which may denature the protein).
A conjugation group may be strongly electrophilic or nucleophilic and thereby capable of reacting directly with a targeting moiety. A precursor to a conjugation group may be a weaker electrophile or nucleophile that requires activation prior to conjugation with a targeting moiety. Conversion of a L group from a precursor group to a conjugation group is generally performed in a separate step prior to conjugation with a targeting moiety. However, where a targeting moiety is unreactive with the conversion reagents and unaffected by the reaction conditions, it is possible to generate a conjugation group in the presence of the targeting moiety.
An electrophilic conjugation group may react directly with a nucleophile, either through nucleophilic substitution or nucleophilic addition. In the present invention, electrophilic conjugation groups react with the targeting moiety acting as the nucleophile. A targeting moiety may naturally possess nucleophilic group(s). For example, a targeting moiety may contain an amino group or a sulfhydryl group. Alternatively, a targeting moiety may have been modified to contain nucleophilic group(s). Procedures for modifying molecules to contain nucleophilic groups are well known to those in the art (see, e.g., catalog of Pierce Chemical Co., Rockford, Ill., and U.S. Pat. No. 4,659,839).
Electrophilic groups which provide conjugation through nucleophilic substitution include those groups which contain substituents which are readily displaced. :Such readily displaced substituents are commonly referred to as leaving groups. Leaving groups include halides which are readily displaced from alkyl halides and alphahalo carbonyl compounds, and carboxylate and stabilized oxyanions which are readily displaced from carbonyl-containing groups such as anhydrides and active esters, respectively. For example, in addition to halide ion leaving groups such as iodide, bromide, and chloride ions, other leaving groups include carboxylate ions such as acetate and trifluoroacetate and phenolate ions such as phenolate and p-nitrophenolate as well as tosylates and mesylates. Suitable active ester groups include N-hydroxysuccinimidyl, tetrafluorophenyl, nitrophenyl, and 1-hydroxybenzotriazolyl.
Electrophilic groups which provide conjugation through nucleophilic addition include those groups which contain unsaturated carbon atoms susceptible to nucleophilic addition. Suitable electrophilic carbon species include thiocyanates, isocyanates, isothiocyanates and maleimides.
As mentioned above, a conjugation group capable of reacting directly with a targeting moiety may be prepared by conversion of a weaker electrophilic or nucleophilic group to a stronger one. For example, acarboxylic acid group is a precursor group which may be activated, (e.g., by conversion into an active ester conjugation group capable of reaction with targeting moieties as described above). Another example of a conversion to a strong electrophilic group is deprotection of a phenylsulfonyl succinimide to provide a maleimide capable of reaction with nucleophilic targeting moieties as described above.
The conjugation group may also be a nucleophilic group, such as an amino or sulfhydryl group. Such a nucleophile is capable of reacting with an electrophilic targeting moiety, such as one that naturally possesses electrophilic group(s) or one that has been modified to include electrophilic group(s) For example, a targeting moiety may contain an active ester or a maleimide group. Alternatively, procedures for modifying molecules to contain electrophilic groups are well known to those in the art (see, e.g., catalog of Pierce Chemical Co., Rockford, Ill., and U.S. Pat. No. 4,671,958).
Alternatively, Z may be a targeting moiety rather than a conjugation group. A xe2x80x9ctargeting moietyxe2x80x9d in the compounds of the present invention has the functional property that it binds to a defined target cell population, such as tumor cells. Preferred targeting moieties useful in this regard include proteins, peptides, antibody and antibody fragments, hormones, and vitamins such as biotin. Proteins corresponding to known cell surface receptors (including low density lipoproteins, transferrin and insulin), fibrinolytic enzymes, anti-HER2, platelet binding proteins such as annexins, avidin, streptavidin, and biological response modifiers (including interleukin, interferon, erythropoietin, colony-stimulating factor, TNF-tissue necrosis factors and similar cytokines) are also preferred targeting moieties. Also, anti-EGF receptor antibodies, which internalize following binding to the receptor and traffic to the nucleus to an extent, are preferred targeting moieties for use in the present invention to facilitate delivery of Auger emitters and nucleus binding drugs to target cell nuclei. Oligonucleotides, e.g., antisense oligonucleotides that are complementary to portions of target cell nucleic acids (DNA or RNA), are also useful as targeting moieties in the practice of the present invention. Oligonucleotides binding to cell surfaces are also useful. Analogs, including those of the above-listed targeting moieties, that retain the capacity to bind to a defined target cell population may also be used within the claimed invention. In addition, synthetic or recombinant targeting moieties may be designed and produced.
Functional equivalents of the aforementioned molecules are also useful as targeting moieties of the present invention. An example of a targeting moiety functional equivalent is a xe2x80x9cmimeticxe2x80x9d compound, which is an organic chemical construct designed to mimic the proper configuration and/or orientation for targeting moiety-target cell binding.
Another example of a targeting moiety functional equivalent is a short polypeptide designated as a xe2x80x9cminimalxe2x80x9d polypeptide. Such a polypeptide is constructed using computer-assisted molecular modeling and mutants having altered binding affinity of the targeting moiety.
As disclosed above, preferred targeting moieties of the present invention are proteins, antibodies (polyclonal or monoclonal), peptides, oligonucleotides or the like. Polyclonal antibodies useful in the practice of the present invention are polyclonal (Vial and Callahan, Univ. Mich. Med. Bull. 20:284-6, 1956), affinity-purified polyclonal or fragments thereof (Chao et al., Res. Comm. in Chem. Path. and Pharm. (:749-61, 1974).
Monoclonal antibodies useful in the practice of the present invention include whole antibody and fragments thereof. Such monoclonal antibodies and fragments are producible in accordance with conventional techniques, such as hybridoma synthesis, recombinant DNA techniques and protein synthesis. Useful monoclonal antibodies and fragments may be derived from any species (including humans) or may be formed as chimeric proteins which employ sequences from more than one species. See, generally, Kohler and Milstein, Nature 256:495-97, 1975; Eur. J. Immunol. 6:511-19, 1976.
Human monoclonal antibodies or xe2x80x9chumanizedxe2x80x9d murine antibody are also useful as targeting moieties in accordance with the present invention. For example, a murine monoclonal antibody may be xe2x80x9chumanizedxe2x80x9d by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e., containing the antigen binding sites) or the complementary determining regions (xe2x80x9cCDR""sxe2x80x9d) thereof with the nucleotide sequence encoding a human constant domain region and an Fc region (i.e., human framework), e.g., in a manner similar to that disclosed in U.S. Pat. Nos. 4,816,39.7, 4,816,567, 5,530,101 and 5,585,089. Some murine residues may also be retained within the human variable region framework domains to ensure proper target site binding characteristics. Humanized targeting moieties are recognized to decrease the immunoreactivity of the antibody or polypeptide in the host recipient, permitting an increase in the half-life and a reduction in the possibility of adverse immune reactions.
Another preferred targeting moiety of the present invention is an annexin and other platelet binding proteins, such as PAP-1 (Placental Anticoagulant Protein or Annexin V). Annexins are (with the exception of annexin II), single-chain, non-gylcosylated protein of approximately 36 kilodaltons. In the presence of calcium, these proteins have an especially high affinity for negatively-charged phospholipids, such as (a phosphatitylserine.
As mentioned above, W is a hydrolyzable group. As used herein, the term xe2x80x9chydrolyzable groupxe2x80x9d refers to any neutral organic group that provides a charged group upon hydrolysis. The hydrolysis may be chemical or enzymatic in nature. Examples of hydrolyzable groups include esters, imidates, and nitrites which may be hydrolyzed to carboxylic acids; and carbamates which may be hydrolyzed to amines.
Referring to the above formula, the distance by which the chelating nitrogen atoms are separated may be increased by interposing a methylene group, xe2x80x94CH2xe2x80x94, between the carbon atoms bonded to the nitrogens depicted. When no methylene group is interposed, represented in the above formula where n=0, the chelating nitrogens are separated by two carbon atoms. When n=1, the interposed methylene group may be substituted with R3.
R3 may be hydrogen, a lower alkyl group, an alkyl group, an alkoxy group, a halogen, a hydroxyl group, a nitro group, xe2x80x94(CH2)mxe2x80x94Z or xe2x80x94(CH 2)mxe2x80x94W. As used throughout, a lower alkyl group is an alkyl group of hydrocarbon radicals having from 1-10 carbon atoms, and physiologically acceptable salts of the acid radicals which includes a substituted lower alkyl. A substituted lower alkyl group is a lower alkyl group that bears a halogen, perhaloalkyl, hydroxyl or alkoxy substituent; an alkoxy group is any alkoxy group of C6 or less. Suitable halogens include fluorine, chlorine, bromine and iodine.
R4 and R5 may be attached at one or more of the aromatic ring positions, preferably the ring carbon atoms. R4 and R5 are independently selected from hydrogen, a lower alkyl group, an alkoxy group, a halogen, a hydroxyl group, a nitro group, xe2x80x94(CH2)mxe2x80x94Z and xe2x80x94(CH2)mxe2x80x94W. For R4 and R5, preferred groups include lower alkyl groups such as methyl, alkoxy groups such as methoxy, and halogen groups such as fluorine. Preferred Z groups include active esters such as N-hydroxysuccinimide esters and maleimides. Preferred W groups include ester and carbamate groups, such as ethyl esters and ethyl carbamates. Preferably, such preferred alkyl groups, alkoxy groups, and ester groups are substituted at the aromatic ring carbon ortho or para to the chelating nitrogen depicted in formula I above.
R6 and R7 may be independently a hydrogen, lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z, xe2x80x94(CH2)mxe2x80x94W and 
where Q represents a multivalent acid functionality capable of coordinating with metal ions, and p=0 to 1; R12 and R13 are independently selected from hydrogen, hydroxyl, carboxyl, phosphonic, and hydrocarbon radicals having from 1-10 carbon atoms, and physiologically acceptable salts of the acid radicals, and R12 and R13 may be the same as or different from one another. In one embodiment, R7 may be hydrogen when R6 is 
and Q is a phosphonic or a carboxylic acid and R6 may be hydrogen when R7 is 
and Q is a phosphonic or a carboxylic acid, but R6 and R7 cannot both be hydrogen simultaneously. As noted above, Q represents a multivalent acid functionality. As used herein, the term multivalent acid functionality refers to any multivalent acid capable of coordinating a metal ion known to one of ordinary skill in the art. In preferred embodiments, R6 or R7 or both bear a Q containing substituent. Preferred multivalent acids are the following: a phosphonic acid, a carboxylic acid, a thioacetic acid and a sulfonic acid. Particularly preferred are a phosphonic acid and a carboxylic acid. The multivalent acid provides extra donor atoms which allow for binding of a metal through coordination of such donor atoms, thereby providing for a versatile chelation compound for diagnostic and therapeutic use.
The compounds of the present invention typically have one or more Q, Z and/or W groups. For example, a compound may have one Z or one W or one Q, or a combination of all three or some lesser combination. Alternatively, for example, a compound may have multiple Z and/or multiple W groups, and/or multiple Q groups.
A and Axe2x80x2 may be independently selected from nitrogen, oxygen and sulfur. Where a sulfur is present, it may bear a hydrogen or a sulfur protecting group. Where A and Axe2x80x2 are both sulfur, they may be joined together by a bond or any sulfur protecting group known in the art. Where an oxygen or a nitrogen is present, it may bear a hydrogen. Where A or Axe2x80x2 is nitrogen, A may bear R8 or R10 or both and Axe2x80x2 may bear R9 or R11 or both, wherein R8, R9, R10 and R11 are independently selected from a lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z, xe2x80x94(CH2)mxe2x80x94W and 
where Q represents a multivalent acid functionality capable of coordinating with metal ions, and p=0 to 1; R12 and R13 are independently selected from hydrogen, hydroxyl, carboxyl, phosphonic, and hydrocarbon radicals having from 1-10 carbon atoms, and physiologically acceptable salts of the acid radicals, and R12 and R13 may be the same as or different from one another; R8 and R10 may be joined to form a cyclic anhydride or R9 and R11 may be joined to form acyclic anhydride. Where A and Axe2x80x2 are both nitrogen, R10 and R11 may be joined to form T, where T is 
and n is 0 to 1. R1 xe2x80x2 and R2xe2x80x2 may be independently selected from hydrogen, xe2x95x90O, xe2x80x94(CH2)mxe2x80x94Z and xe2x80x94(CH2)mxe2x80x94W, or R1xe2x80x2 and R2xe2x80x2 are taken together to form a cyclic la anhydride or a benzene ring. R3xe2x80x2 is selected from hydrogen, lower alkyl, substituted lower, alkoxy, perhaloalkoxy, perhaloalkyl, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z and xe2x80x94(CH2)mxe2x80x94W. In a preferred embodiment where A and Axe2x80x2 are both sulfur, the sulfur atoms are joined together by a bond thus forming a disulfide. In a preferred embodiment where A and Axe2x80x2 are both nitrogen, R10 and R11 are joined to form T where n is either 0 or 1 and R8 and R9 are 
where Q represents multivalent acid functionality capable of coordinating with metal ions, and m=0 to 1; R12 and R13 are independently selected from hydrogen, hydroxyl, carboxyl, phosphonic, and hydrocarbon radicals having from 1-10 carbon atoms, and physiologically acceptable salts of the acid radicals, and R12 and R13 may be the same as or different from one another.
The chelation compounds of the present invention may be categorized by the number and type of.chelating atoms (i.e. NxSyOz where x is 2 to 4, y is 0 to 2, and z is 0 to 2). For example, where both A and Axe2x80x2 are nitrogen, the chelation compounds of the present invention are able to bind a metal through coordination with all four nitrogen atoms. Such a chelating compound may be referred to as an xe2x80x9cN4xe2x80x9d (N4S0O0) compound. In another embodiment, both A and Axe2x80x2 are sulfur, resulting in the capacity for metal chelation through two nitrogen atoms and two sulfur atoms, and thus providing an xe2x80x9cN2S2xe2x80x9d (N2S2O0) chelating compound. Alternatively, A may be nitrogen and Axe2x80x2 may be sulfur or A may be sulfur and Axe2x80x2 may be nitrogen. Either of these embodiments are capable of metal chelation involving three nitrogen atoms and a single sulfur atom, an xe2x80x9cN3Sxe2x80x9d (N3S1O0) chelating compound. In another embodiment, A and/or Axe2x80x2 may be oxygen atoms (e.g., hydroxyl groups). Where both A and Axe2x80x2 are oxygen, an xe2x80x9cN2O2xe2x80x9d (N2S0O2) chelating compound results. Other embodiments include xe2x80x9cN3Oxe2x80x9d (N3S0O1) and xe2x80x9cN2SOxe2x80x9d (N2S1O1) chelation compounds where one of either A or Axe2x80x2 is oxygen and the other is nitrogen or sulfur, respectively.
In a preferred embodiment of the present invention, the chelation compounds are able to bind a metal radionuclide with the donor atoms providing up to eight coordination sites. For example, A and Axe2x80x2 are both nitrogen, R10 and R11 may join the two nitrogen atoms, through the formation of T to create a cyclic xe2x80x9cN4xe2x80x9d (N4S0O0) chelation compound and wherein R6, R7, R8 and R9 may be 
wherein Q is preferably a multivalent acid functionality, such as a phosphonic and/or a carboxylic acid. Thus, in addition to the four chelating atoms of nitrogen, the oxygen atoms of the multivalent acid functionality provide up to four additional coordination sites, thereby expanding the type of radionuclide that is useful in this invention, (e.g., Indium and Yttrium).
As noted above, the sulfur atoms of the chelation compounds may bear sulfur protecting groups. Suitable sulfur protecting groups include any of the alkyl, acyl, and aryl groups, disulfides and bunte salts known by those of ordinary skill in the art. Preferred sulfur protecting groups are those that result in the formation of thioacetal, hemithioacetal, thioketal, hemithioketal, thioester or acetamidomethyl substituent. Particularly preferred groups include p-anisylidine, acetonyl, tetrahydrylfuranyl, ethoxyethyl, tetrahydrylpyranyl, acetamidomethyl and derivatives thereof. When conjugating a chelating compound of the present invention to a targeting moiety, the protecting groups may be-removed just prior to metal complexation or during the radiolabeling reaction.
An acetamidomethyl sulfur-protecting group is represented by the following formula, wherein the sulfur atom shown is a sulfur donor atom of the chelating compound: 
The acetamidomethyl group is displaced from the chelating compound during radiolabeling conducted at about 50xc2x0 C. in a reaction mixture having a pH of about 3 to 6.
When hemithioacetal protective groups are used, each sulfur atom to be protected has a separate protective group attached to it, which together with the sulfur atom defines a hemithioacetal group. The hemithioacetal groups contain a carbon atom bonded directly (i.e., without any intervening atoms) to a sulfur atom and an oxygen atom, i.e., 
Preferred hemithioacetals generally are of the following formula, wherein the sulfur atom is a sulfur atom of the chelating compound, and a separate protecting group is attached to each of the sulfur atoms on the chelating compound: 
wherein Ra is a lower alkyl group, preferably of from 2-5 carbon atoms, and Rb is a lower alkyl group, preferably of from 1-3 carbon atoms. Alternatively, Ra and Rb may be taken together with the carbon atom and the oxygen atomxe2x80x2 shown in the formula to define a nonaromatic ring, preferably comprising from 3-7 carbon atoms in addition to the carbon and oxygen. atoms shown in the formula. Rc represents hydrogen or a lower alkyl group wherein the alkyl group preferably is of from 1-3 carbon atoms. Examples of such preferred, hemithioacetals include, but are not limited to: 
In one embodiment of the present invention, the sulfur protecting groups may join the two sulfur chelating atoms. Preferred embodiments of the sulfur protecting groups include thioketals and thioacetals, which may be prepared by condensation of the sulfur containing chelating compound with ketones and aldehydesi respectively. These particular sulfur protecting groups are represented by the following formula, wherein the sulfur atoms shown are the sulfur donor atom of the chelating compound: 
In the formula, Rd and Re are independently selected from hydrogen, lower alkyl groups (preferably methyl or ethyl), lower alkoxy groups (preferably containing one or two carbon atoms), aryl groups, or taken together form a cyclic group (preferably a cyclopentane or cyclohexane ring).
These sulfur protective groups are displaced during the radiolabeling reaction, conducted at acidic pH, in what is believed to be metal-assisted acid cleavage. Covalent bonds form between the sulfur atoms and the metal radionuclide. A separate step for removal of the sulfur-protective groups is not necessary. The radiolabeling procedure thus is simplified. In addition, the basic pH conditions and harsh conditions associated with certain known radiolabeling procedures or procedures for removal of other sulfur protective groups are avoided. Thus, base-sensitive groups on the chelating compound survive the radiolabeling step intact. Such base labile groups include any group which may be destroyed, hydrolyzed, or otherwise adversely affected by exposure to basic pH. In general, such groups include esters, maleimides, and isothiocyanates, among others. Such groups may be present on the chelating compound as conjugation groups.
The aromatic ring atoms designated as X, Y, Xxe2x80x2 and Yxe2x80x2 are independently selected from carbon, nitrogen, sulfur and oxygen to independently form five or six member rings, wherein the remaining ring atoms are carbon. The aromatic rings containing X and Y or Xxe2x80x2 and Yxe2x80x2 are selected independently. For example, one ring may be a five member ring and the other a six member ring. For six member rings, where X, Y, Xxe2x80x2 and Yxe2x80x2 are all carbon, the aromatic rings are benzene type rings. Where X, Y, Xxe2x80x2 and Yxe2x80x2 are all nitrogen, the aromatic rings are pyrimidine type rings. Where one of X or Y and one of Xxe2x80x2 or Yxe2x80x2 are nitrogen, the aromatic rings are pyridine type rings.
For five member rings where X, Y, Xxe2x80x2 and Yxe2x80x2 are all nitrogen, the aromatic rings are imidazole or pyrazole type rings. Where one of X or Y and one of Xxe2x80x2 or Yxe2x80x2 are sulfur, the aromatic rings are thiophene type rings. Where one of X or Y and one of Xxe2x80x2 or Yxe2x80x2 are sulfur and nitrogen, the aromatic rings are thiazole or isothiazole type rings, where on of X or Y and one of Xxe2x80x2 or Yxe2x80x2 are oxygen, the aromatic rings are furan type rings, where one of X or Y and one of Xxe2x80x2 or Yxe2x80x2 are oxygen and nitrogen, the aromatic rings are oxazole or isoxazole type rings.
Preferred embodiments of the aromatic rings designated X, Y, Xxe2x80x2 and Yxe2x80x2 include benzene, pyrimidine, pyridine and thiophene, the most preferred being benzene or thiophene. These particular aromatic rings are interchangeable within the chelating compound formula because they are either structurally related or contribute similar properties, e.g., spatial configuration, electronic resonance and inductive properties (i.e., electron withdrawing and donating effects).
The chelation compounds and metal chelates of the present invention may also be asymmetric with respect to the nature of the aromatic rings. For example, the aromatic rings are a combination of benzene and pyridine types where X and Y are both carbon and either Xxe2x80x2 or Yxe2x80x2 are both carbon, or either X or Y is nitrogen and Xxe2x80x2 and Yxe2x80x2 is nitrogen, or either X or Y is nitrogen and Xxe2x80x2 and Yxe2x80x2 are both carbon. In another embodiment, the aromatic rings are a combination of benzene and pyrimidine types where X and Y are both carbon and Xxe2x80x2 and Yxe2x80x2 are both nitrogen, or X and Y are both nitrogen and Xxe2x80x2 and Yxe2x80x2 are both carbon. In another embodiment, the aromatic rings are a combination of pyridine and pyrimidine types where either X or Y is nitrogen and Xxe2x80x2 and Yxe2x80x2 are both nitrogen, or X and Y are both nitrogen and either Xxe2x80x2 or Yxe2x80x2 are nitrogen. In another embodiment, the aromatic rings are a combination of benzene and thiophene types where either X or Y are both carbon and either Xxe2x80x2 or Yxe2x80x2, is sulfur, or either X or Y is sulfur and Xxe2x80x2 and Yxe2x80x2 are both carbon. In another embodiment, the aromatic rings are a combination of pyridine and thiophene types whether either X or Y is nitrogen and one of which is carbon and Xxe2x80x2 or Yxe2x80x2 is sulfur and one of which is carbon, or either X or Y is sulfur, one of which is carbon and either Xxe2x80x2 or Yxe2x80x2 is nitrogen, one of which is carbon. Further variations of the aromatic rings of the presently identified chelation compounds will be evident to one of ordinary skill in the based on the present disclosure in view of the art.
As noted above, in addition to providing chelation compounds, the present invention provides radionuclide metal chelate compounds wherein a metal is chelated (complexed). The chelation compounds of the present invention rapidly form stable metal complexes (radionuclide metal chelates) when reacted with a metal.
The preferred radionuclide metal chelate compound (complexes) of the present invention have the formula (II): 
wherein R1-R11, n, X, Xxe2x80x2, Y, Yxe2x80x2 are described above. A and Axe2x80x2 may be independently selected from nitrogen, sulfur and oxygen. M is a radiometal or a radionuclide metal oxide or nitride, capable of being chelated by a compound of the present invention. Preferred metals and metal oxides or nitrides include radionuclides of copper, yttrium, ruthenium, technetium, rhodium, palladium, gadolinium, samarian, holmium, ytterbium, lutetium, indium, rhenium, gold, lead and bismuth. Particularly preferred are 64Cu, 67Cu, 90Y, 97Ru, 99mTc, 105Rh, 109Pd, 111In, 153Sm, 159Gd, 166Ho, 175Yb, 177Lu, 186Re, 188Re, 198Au, 199Au, 203Pb, 212Pb and 212Bi.
Methods for preparing these isotopes are known. Molybdenum/technetium generators for producing 99mTc are commercially available. Procedures for producing 186Re include the procedures described by Deutsch et al. (Nucl. Med. Biol. 13(4):465-477, 1986) and Vanderheyden et al. (Inorganic Chemistry 24:1666-1673, 1985) (see also U.S. Pat. No. 5,053,186), and methods for production of 188Re have been described by Blachot et al. (Intl. J. of Applied Radiation and Isotopes 20:467-470, 1969) and by Klofutar et al. (J. of Radioanalytical Chem. 5:3-10, 1970) (see also U.S. Pat. No. 4,859,431). Production of 109Pd is described in Fawwaz et al. (J. Nucl. Med. 25:786, 1984). Production of 212Pb and 212Bi is described in Gansow et al. (Amer. Chem. Soc. Symp. Ser 241:215-217, 1984) and Kozah et al. (Proc. Natl. Acad. Sci. USA 83:474-478, 1986). Production of 90Y, a particle emitting therapeutic radionuclide resulting from transmutation processes (without non-radioactive carrier forms present), is commercially available from several sources, including Pacific ixe2x80x2 Northwest National Laboratory, located in Richland, Wash.; Nordion International Inc., locatedin Kanata, Ontario, Canada and by Du Pont as NEN Research products located in North Billerica, Massachusetts. Production of 153Sm is described in Goeckeler et al (Nucl. Med. Biol., Vol. 20, No. 5, pp. 657-661, 1993). 111In is commercially available as INDICLOR(trademark) supplied by Amersham Healthcare located in Arlington Heights, Ill. 99mTc is preferred for diagnostic use, and the other radionuclides listed above are preferred for therapeutic use.
In one embodiment of the present invention, chelation compounds of the invention.including acetamidomethyl and/or hemithioacetal sulfur protective groups are radiolabeled with a metal radionuclide by reacting the compound with the radionuclide under conditions of acidic pH. It is believed that the acidic pH and the presence of the metal both contribute to the displacement of the sulfur protective groups from the chelating compound. The radionuclide is in chelatable form when reacted with the chelation compounds of the invention.
In the case of technetium and rhenium being in xe2x80x9cchelatable formxe2x80x9d generally requires a reducing step. A reducing agent will be employed to reduce the radionuclides (e.g., in the form of pertechnetate and perrhenate, respectively) to a lower oxidation state at which chelation will occur. Many suitable reducing agents, and the use thereof, are known . (See, for example, U.S. Pat. Nos. 4,440,738; 4,434,151; and 4,652,440.) Such reducing agents include stannous ion (e.g., in the form of stannous salts such as stannous chloride or stannous fluoride), metallic tin, ferrous ion (e.g., in the form of ferrous salts such as ferrous chloride, ferrous sulfate, or ferrous ascorbate) and many others. Sodium pertechnetate (i.e., 9mTcO4xe2x88x921 which is in the +7 oxidation level) or sodium perrhenate (i.e., 188ReO4xe2x88x921, 186ReO4xe2x88x921) may be combined simultaneously with a reducing agent and a chelating compound of the invention, in accordance with the radiolabeling method of the invention, to form a chelate.
Preferably, the radionuclide is treated with a reducing agent and a complexing agent to form an intermediate complex (i.e., an xe2x80x9cexchange complexxe2x80x9d). Complexing agents are compounds which bind the radionuclide more weakly than do the chelate compounds of the invention, and may be weak chelators. Any of the suitable known complexing agents may be used, including gluconic acid, glucoheptonic acid, tontanic acid, methylene disphosphonate, glyceric acid, glycolic acid, mannitol, oxalic acid, malonic acid, succinic acid, bicine, N,Nxe2x80x2-bis(2-hydroxy ethyl) ethylene diamine, citric acid, ascorbic acid and gentisic acid. Good results are obtained using gluconic acid or glucoheptonic acid as the Tc-complexing agent and citric acid for rhenium. When the radionuclide in the form of such an exchange complex is reacted with the chelation compounds of the invention, the radionuclide will transfer to the chelation compounds, which bind the radionuclide more strongly to form chelates of the invention. In some instances, heating is necessary to promote transfer of the radionuclide. Radionuclides in the form of such complexes also are considered to be in xe2x80x9cchelatable formxe2x80x9d for the purposes of the present invention.
Y-90 is a particularly preferred radionuclide for therapy, because it exhibits favorable nuclear properties including high specific activity, long path length with respect to deposition of radiation in tissue, high equilibrium dose constant and favorable half-life properties. More specifically, the beta emission of Y-90 (Betaav=0.937 MeV) makes it one of the most energetic of all beta emitters. The X90 value of Y-90 is 5.34 mm (i.e., 90% of the energy emitted from a point source is absorbed in a sphere of 5.34 mm radius). Y-90 has a high equilibrium dose constant or mean energy/nuclear transition, Delta=1.99 Rad-gram/microcurie-hour, and a 64 hour half-life suitable for targeted therapy. Y-90 can be manufactured at high specific activity and is available as a generator product. Specific advantages of Y-90 are (1) that it has the capability to kill neighboring target cells not directly targeted by conventional methods (2) that more radiation is deposited per microcurie localized than for other beta emitters of lower mean particle energy (provided that a sufficiently large target volume is available).
Chelates of 212Pb, 212Bi, 109 Pd, Cu64 and Cu67 may be prepared by combining the appropriate salt of the radionuclide with the chelating compound and incubating the reaction mixture at room temperature or at higher temperatures. It is not necessary to treat the lead, bismuth, palladium and copper radioisotopes with a reducing agent prior to chelation, as such isotopes are already in an oxidation state suitable for chelation (i.e., in chelatable form). The specific radiolabeling reaction conditions may vary somewhat according to the particular radionuclide and chelating compound involved.
In another embodiment of the present invention, where the sulfurs are protected by formation of a disulfide bond, chelation compounds of the invention are radiolabeled following reduction of the disulfide bond under mild conditions. For example, the disulfide may be reduced with SnCl2 under conditions which do not reduce disulfides on proteins such as antibodies.
The chelation compounds and metal chelates of the present invention have a variety of uses, although certain uses are preferred depending upon the particular embodiment. In one embodiment of the present invention, the chelation compounds can be employed in the pretargeting methods as described in U.S. Pat. No. 5,608,060.
In another embodiment of the present invention, the chelation compounds and the radionuclide metal chelates are either reactive with a targeting moiety, or are conjugated to a targeting moiety. These compounds may be generally represented by the above-described compounds which bear the group Z. A chelating compound or a metal chelate that is reactive with a targeting moiety bears at least one conjugation group Z. Such conjugation groups include those described above (e.g., an active ester or a maleimide). Alternatively, the chelating compound or metal chelate may be conjugated to a targeting moiety Z. Such targeting moieties include those described above (e.g., proteins and antibodies). The preparation of representative chelation compounds that are reactive with targeting moieties is presented in the examples below. The preparation of representative radionuclide metal-targeting moiety conjugates is also presented in the examples below.
In the practice of the present invention, metal chelate-targeting moiety conjugates may be prepared by complexation of the radionuclide metal either before or after the chelating compound is conjugated to the targeting moiety. More specifically, a conjugate may be xe2x80x9cpre-formedxe2x80x9d or xe2x80x9cpost-formed,xe2x80x9d depending upon whether the chelating compound and targeting moiety are joined after or before the complexation of the radionuclide metal. A pre-formed conjugate comprises a chelating compound of the present invention that is first labeled with a radionuclide metal and then is conjugated to a targeting moiety. A post-formed conjugate comprises a chelating compound of the present invention that is first conjugated to a targeting moiety and then is labeled with a radionuclide metal. Thus, for pre-formed conjugates, the radionuclide is added to the chelating compound prior to the addition of the targeting moiety, whereas, for post-formed conjugates, the radionuclide is added after the addition of the targeting moiety. The final conjugate is the same regardless of how formed.
Generally, the chelation compounds of the present invention that are either reactive with targeting moieties or are conjugated to targeting moieties may be represented by the formula (I) above, where the specific embodiments of the elements of the formula include the following:
R1 and R2 may be independently hydrogen (H), an oxy group (xe2x95x90O); or xe2x80x94(CH2)mxe2x80x94Z where m is 0-10 and Z represents a conjugation group or targeting moiety; or R1 and R2 may be taken together to form a cyclic anhydride or a benzene ring.
The distance between the chelating nitrogen atoms of formula (I) may be varied by the imposition of a methylene group. When imposed, the methylene group may be substituted with R3.
R3 may be hydrogen, a lower alkyl group, an alkoxy group, a halogen, a hydroxyl group, a nitro group, or xe2x80x94(CH2)mxe2x80x94Z.
R4 and R5 may be attached at one or more of the aromatic ring positions, preferably the ring carbon atoms, and are independently selected from hydrogen, a lower alkyl group, an alkoxy group, a halogen, a hydroxyl group, a nitro group, and xe2x80x94(CH2)mxe2x80x94Z.
R6 and R7 are independently selected from lower alkyl, alkoxy, halogen, hydroxyl, nitro, xe2x80x94(CH2)mxe2x80x94Z, and 
where Q represents multivaleht acid functionality capable of coordinating with metal ions, and m=0 to 1; R12 and R13 are independently selected from hydrogen, hydroxyl, carboxyl, phosphonic, and hydrocarbon radicals having from 1-10 carbon atoms, and physiologically acceptable salts of the acid radicals, and R12 and R13 may be the same as or different from one another.
The chelation compounds reactive with or conjugated to targeting moieties have at least one Z, but may contain more than one Z. For example, any two groups selected from R1-R5 may be Z.
A, Axe2x80x2, X, Xxe2x80x2, Y, Yxe2x80x2 and n are as described above for formula (I).
Similarly, the radionuclide metal chelate compounds of the present invention that are either reactive with targeting moieties or are conjugated to targeting moieties may be represented by the formula (II). The specific embodiments of those elements of the formula denoted by R1-R5, n, X, Xxe2x80x2, Y, and Yxe2x80x2 are as described immediately above for the chelation compounds. M is a radionuclide; radionuclide metal oxide or radionuclide metal nitride. The metal chelate compounds reactive with or conjugated to targeting moieties have at least one Z, but may contain more than one Z.
In a preferred embodiment, the compounds of the present invention are xe2x80x9cN4xe2x80x9d (N4S0O0) chelation compounds and metal chelates. Therefore, for preferred chelation compounds and metal chelates, A and Axe2x80x2 are nitrogen. For particularly preferred chelation compounds, A and Axe2x80x2 are nitrogen atoms joined together by a bond, i.e., R10 and R11 form T, the chelation compounds are tetraazacyclic, a tetradectane system. Preferred compounds of the present invention have X, Y, Xxe2x80x2 and Yxe2x80x2 as carbon, nitrogen and sulfur. For the metal chelates of the present invention, technetium (e.g., 99mTc) and indium (e.g., 111In) are the preferred metals for diagnostic purposes, and rhenium (e.g., 186Re and 188Re) and yttrium (e.g., 90Y) are the preferred metals for therapeutic purposes.
Further, in a preferred embodiment, the compounds of the present invention, which are reactive with targeting moieties, possess a single conjugation group. A preferred conjugation group is the N-hydroxysuccinimide ester group.
In a preferred embodiment, in addition to the above-mentioned preferences, the conjugation group is an aromatic ring substituent, i.e., either R4 or R5 is xe2x80x94(CH2)mxe2x80x94Z. For one such preferred embodiment, n=1, R1-R4 are hydrogen, and R5 is xe2x80x94(CH2)mxe2x80x94Z, where m=0 and Z is an active ester such as an N-hydroxysuccinimide ester. Alternatively, the conjugation group may be a substituent of the carbons linking the chelating nitrogens, i.e.; R1-R3. In one such preferred embodiment, n=1, R1 or R2 are xe2x80x94(CH2)mxe2x80x94Z where m=0 and Z is an N-hydroxysuccinimide ester, R3 is hydrogen, and R4 and R5 are hydrogen. In another such preferred embodiment, n=1, R1 and R2 are hydrogen, R3 is xe2x80x94(CH2)mxe2x80x94Z as described immediately above, and R4 and R5 are hydrogen. R6, R7, R8 and R9 may be 
where Q represents multivalent acid functionality capable of coordinating with metal ions, and m=0 to 1; R12 and R13 are independently selected from hydrogen, hydroxyl, carboxyl, phosphonic, and hydrocarbon radicals having from 1-10 carbon atoms, and physiologically acceptable salts of the acid radicals, and R12 and R13may be the same as or different from one another.
In another preferred embodiment, in the xe2x80x9cN4xe2x80x9d (N4S0O0) compounds of the present invention the conjugation group is an anhydride, i.e., R8 and R10 and R9 and R11 are taken together in a vicinyl configuration, to form a cyclic anhydride, xe2x80x94(CH2)mxe2x80x94Z, where m=1 and Z is a carboxylic acid anhydride resulting from vicinyl dicarboxylic acids. In one such embodiment, in addition to the above-mentioned preferences, R1-R5 are hydrogen, n=1, R6 and R7 are 
where Q represents multivalent acid functionality capable of coordinating with metal ions, and p=0 to 1; R12 and R13 are independently selected from hydrogen, hydroxyl; carboxyl, phosphonic, and hydrocarbon radicals having from 1-10 carbon atoms, and physiologically acceptable salts of the acid radicals, and R12 and R13 may be the same as or different from one another.
In another preferred embodiment, the conjugation group is an anhydride, i.e., R1 and R2 are taken together to form a cyclic anhydride. In one such embodiment, in addition to the above-mentioned preferences, R1 and R2 are taken together to form a cyclic anhydride, n=0, and R4 and R5 are fluorine.
For the compounds of the present invention which are conjugated to targeting moieties, preferred targeting moieties include proteins such as antibodies and annexin as well as binding protens such as avidin and streptavidin.
In another aspect of the present invention, the chelation compounds and the radionuclide metal chelate compounds are used in radiopharmaceutical applications without the necessity for a conjugation group or targeting moiety. Such chelating and metal chelate compounds are useful by virtue of their lipophilic properties and may be generally represented by the above-described compounds which bear hydrolyzable group W.
Generally, the chelation compounds of the present invention that are useful without possessing a conjugating group or targeting moiety may be represented by the formula (I) above where the specific embodiments of the elements of the formula include the following.
R1 and R2 may be independently hydrogen (H), an oxy group (xe2x95x90O); or xe2x80x94(CH2)mxe2x80x94W where W represents a hydrolyzable group; or R1 and R2 may be taken together to form a cyclic anhydride or benzene ring.
The distance between the chelating nitrogen atoms of formula (I) may be varied the imposition of a methylene group, xe2x80x94CH2. When imposed, the methylene group may be substituted with R3.
R3 may be hydrogen, a lower alkyl group, an alkoxy group, a halogen, a hydroxyl group, a nitro group, and xe2x80x94(CH2)mxe2x80x94W.
R4 and R5 may be attached at one or more of the aromatic ring positions, preferably the ring carbon atoms, and are independently selected from hydrogen, a lower alkyl group, an alkoxy group, a halogen, a hydroxyl group, a nitro group, or xe2x80x94(CH2)mxe2x80x94W.
The chelation compounds that are useful in the absence of a conjugation group of targeting moiety have at least one W, but may contain more than one W. For example, any two groups selected from R1-R5 may be W.
A, Axe2x80x2, X, Xxe2x80x2, Y, Yxe2x80x2, R6, R7, and R8 through R11 and n are as described above for formula (I).
Similarly, the radionuclide metal chelate compounds of the present invention that are useful without a conjugation group or targeting moiety may be represented by the formula (II) where the specific embodiments of the elements of the formula, R1-R5, R6-R11, n, X, Xxe2x80x2, Y and Yxe2x80x2 are as described immediately above for the chelation compounds. M is a radionuclide, radionuclide metal oxide or radionuclide metal nitride. The metal chelates that are useful in the absence of a conjugation group or targeting moiety have at least one W, but may contain more than one W.
In a preferred embodiment, W is an enzyme hydrolyzable group, such as an ester or a carbamate. Such groups are subject to hydrolysis by esterases commonly found in tissues such as the heart and bone marrow. In a particularly preferred embodiment, the hydrolyzable group is an ethyl ester or ethyl carbamate.
Preferred embodiments of the compounds which possess hydrolyzable groups W include the preferences for M, A, Axe2x80x2, X, Y, Xxe2x80x2 and Yxe2x80x2 described above for the compounds which possess a conjugation group or a targeting moiety, Z. In a preferred embodiment, the compounds of the present invention having hydrolyzable groups W possess more than one W.
In one preferred embodiment, in addition to the above-mentioned preferences, the hydrolyzable group is an aromatic ring substituent, i.e., R4 and R5 are xe2x80x94(CH2)mxe2x80x94W. For one such embodiment, n=1, R1-R3 are hydrogen, and R4 and R5 are xe2x80x94(CH2)mxe2x80x94W, where m=0 and W is either an ester (i.e., xe2x80x94CO2Et), a carbamate (i.e., xe2x80x94NHxe2x80x94CO2Et) or a nitrile (xe2x80x94CN). Alternatively, in another preferred embodiment, where both R4 and R5 are xe2x80x94(CH2)mxe2x80x94W as described immediately above, n=1, either R1 or R2 is an oxy group (xe2x95x90O) and R3 is either hydrogen or xe2x80x94(CH2)mxe2x80x94W.
In another preferred embodiment, the hydrolyzable group W is a substituent of the carbon atoms linking the chelating nitrogens, i.e., one or more of R1-R3 is xe2x80x94(CH2)mxe2x80x94W. For example, in one such preferred embodiment, in addition to the above noted preference, n=0, R1 and R2 are xe2x80x94(CH2)mxe2x80x94W where m=0 and W is an ester, and R4 and R5 are fluorine. In another such preferred embodiment, n=1, either R1 or R2 is an oxy group (xe2x95x90O), R3 is xe2x80x94(CH2)mxe2x80x94W as described immediately above, and R4 and R5 are methyl. In a further such preferred embodiment, n=1, R1 and R2 are hydrogen, R3 is xe2x80x94(CH2)mxe2x80x94W as described above, and R4 and R5 are methoxy.
The lipophilic properties of these chelating and metal chelate compounds are due in part to the hydrophobic nature of hydrolyzable W. As noted above, W includes any neutral organic group that provides a charged group upon hydrolysis. Generally, the neutral organic group of W is hydrophobic and imparts lipophilic character to the chelating and metal chelate compounds.
The lipophilic compounds of the present invention are particularly useful in vivo where it is desirous to accumulate the metal chelates in tissues such as the heart and bone marrow. In such applications, the administered lipophilic metal chelates reach these tissues through the bloodstream and, because of their lipophilic properties, the metal chelates are absorbed by these tissues. Once absorbed into the tissues, the metal chelates are subject to hydrolysis where the hydrolyzable group, W (e.g., an ester), which imparted lipophilicity to the chelate is converted to a charged species (e.g., an acid if the ester is a carboxylate ester, and a base if the ester is a carbamate ester) and is thereby prevented from escaping the tissue.
Suitable hydrolyzable groups W included nitrites, carbamates, and esters. Preferred hydrolyzable groups include carbamates and carboxylate esters. Preferred carboxylate esters include methyl, ethyl, propyl and isopropyl esters. Preferred carbamate esters include methyl and ethyl esters.
The lipophilic metal chelates of the present invention, which bear hydrolyzable groups W, may undergo either chemical or enzymatic hydrolysis to yield residually charged metal chelates. To be effective, the metal chelates are resistant to rapid hydrolysis in the bloodstream, but are readily hydrolyzed upon uptake by the tissue of interest. Hydrolysis which occurs in the bloodstream is primarily chemical in nature while tissue hydrolysis is primarily enzymatic.
In one embodiment, the compounds of the present invention are additionally resistant toward chemical hydrolysis. For example, the chelation compounds and metal chelates that bear ester groups, which are directly conjugated to the aromatic ring as either ortho or para substituents relative to the chelating nitrogen, are particularly stable toward chemical hydrolysis. Referring to the above formulas, these preferred compounds are represented by those compounds where R4 and/or R5 are xe2x80x94(CH2)mxe2x80x94W (m=0 and W is an ester), and where R4 and/or R5 is located ortho or para to the chelating nitrogen.
Such suitably substituted esters are resistant toward chemical hydrolysis by virtue of electron donation from the chelating nitrogen through the aromatic ring to the ester carbonyl group. This dispersal of electron density renders the ester carbonyl relatively electron rich and reduces its reactivity as an electrophile. Because the rate-determining step in ester hydrolysis is the addition of a nucleophilic water molecule to the ester carbonyl, ester carbonyl groups that are less electrophilic react more slowly toward nucleophilic addition. Thus, ester carbonyl groups which are stabilized toward nucleophilic addition by electron donating groups are resistant toward hydrolysis. For these reasons, the above-described esters of the present invention are resistant toward chemical hydrolysis in the bloodstream.
While the efficacy of the administration of the lipophilic compounds of the present invention resides in part in their stability toward hydrolysis in-the bloodstream, their ultimate utility as radiopharmaceutical agents relies on their capacity to be taken up and retained by various tissues. The uptake of these compounds into the tissue results from the particular character of the compounds and the permeability of the tissues toward such compounds.
The compounds of the present invention are retained within a tissue, such as malignant cells, by conversion of the lipophilic compounds to charged compounds (ionic species) by hydrolysis. The compounds of the present invention, which are resistant to chemical hydrolysis, are readily susceptible to enzymatic hydrolysis. Suitable hydrolyzable groups that are converted to charged compounds by enzymatic action include ester and carbamate groups which are converted to carboxylic acid and amino groups, respectively.
The compounds of the present invention may be taken up by various tissues, but are primarily intended for the tissues containing malignant cells and activated platelets. The metal chelates of the present invention may be selectively taken up by either malignant cell tissue depending upon the nature of the chelate.
The radiolabeled chelates of the present invention have use in diagnostic and therapeutic procedures, both for in vitro assays and for in vivo medical procedures. The radiolabeled chelates may be delivered (e.g., administered to a warm-blooded animal such as a human) intravenously, intraperitoneally, intralymphatically, locally, or by other suitable means, depending on such factors as the type of target site. The amount to be provided will vary according to such factors as the type of radionuclide (e.g., whether it is a diagnostic or therapeutic radionuclide), the route of delivery, the type of target site(s), the affinity of the targeting moiety, if employed, for the target site of interest, and any cross-reactivity of the targeting moiety, if employed, with normal tissues. Appropriate amounts may be established by conventional procedures, and a physician skilled in the field to which this invention pertains will be able to determine a suitable amount for a patient. A diagnostically effective dosage is generally from about 5 to about 35 and typically from about 10 to about 30 mCi per 70 kg body weight. A therapeutically effective dosage is generally from about 20 mCi to about 300 mCi or higher. For diagnosis, conventional non-invasive procedures (e.g., gamma cameras) are used to detect the biodistribution of the diagnostic radionuclide, thereby determining the presence or absences of the target sites of interest (e.g., tumors, heart, brain).
The comparatively low intestinal localization of the therapeutic radiolabeled chelates of the present invention or catabolites thereof permits increased dosages, since intestinal tissues are exposed to less radiation. The clarity and accuracy of diagnostic images also is improved by the reduced localization of radiolabeled chelates or catabolites thereof in normal tissues via an increase in target to nontarget ratio.